Methods and apparatus for the high through-put detection of binding interactions in vivo

ABSTRACT

An apparatus for screening for translocation of a first protein of interest in vivo in a plurality of cells comprises (a) a thin unitary total internal reflection member having a surface portion, (b) a plurality cell contacted to said surface portion by the plasma membrane of said cell, said cell containing said first protein of interest, the protein of interest having a fluorescent group conjugated thereto; (c) a light source operatively associated with the total internal reflection member and positioned for directing a source light into the member to produce an evanescent field adjacent the surface portion, with the evanescent field extending into a first portion of the cell adjacent the plasma membrane, the evanescent field being weaker in a second portion of the cell, the fluorescent group emitting light when in the first portion of the cell and emitting less light when in the second portion of the cell; (d) coupling means for coupling the light source to the thin unitary total internal reflection member and illuminate at least 10 square millimeters of the surface portion; and (e) a light detector operatively associated with the total internal reflection member and configured to detect emitted light from the cells, whereby the emission of more or less light from the cell indicates the translocation of the protein between the first and second portions of the cell.

RELATED APPLICATIONS

This application claims the benefit of provisional application serialNo. 60/216,282, filed Jul. 6, 2000, the disclosure of which isincorporated by reference herein in its entirety, and this applicationis also a continuation-in-part of PCT application PCT/US99/19696, filedAug. 31, 1999 which was published in English under Article 21(2), and inturn claims the benefit of provisional application No. 60/103,092, filedOct. 5, 1998, the disclosures of both of which are incorporated byreference herein in their entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under NationalInstitutes of Health grants RO1GM-51457, R21CA-83229, GM-48113,GM-51457, and 1F32NS10767. The Government has certain rights to thisinvention.

FIELD OF THE INVENTION

The present invention concerns method and apparatus for detectingbinding interactions in cells. The methods and apparatus areparticularly suitable for the high through-put screening of cell arraysand combinatorial libraries.

BACKGROUND OF THE INVENTION

A significant fraction of known cellular signaling proteins have beenshown to translocate to or dissociate from the plasma membrane as partof their activation cycle. In particular, the recruitment of cytosolicproteins by activated receptors and plasma membrane signaling proteinsis a general principle in receptor-mediated signal transduction (Pawson,T. (1995) Nature 373, 573-580; Ullrich, A. & Schlessinger, J. (1990)Cell 61, 203-212; Pfister, et al. (1985) Science 228, 891-893; Hunter,T. (1987) Cell 50, 823-829; Pawson, T. & Scott, J. D. (1997) Science278, 2075-2080). Translocation is often transient with active signalingcomponents dissociating from the plasma membrane and acting on cytosolicand nuclear targets. Recruitment processes are exemplified by thebinding of cytosolic SH2-domain containing proteins to tyrosinephosphorylated plasma membrane receptors (Koch, et al. (1991) Science252, 668-674; Kypta, et al. (1990) Cell 62, 481-492) and by the bindingof cytosolic signaling enzymes to GTP bound small G-proteins at theplasma membrane (Moodie, et al. (1993) Science 260, 1658-1661; Stokoe,et al. Science 264, 1463-1467; Leevers, et al. (1994) Nature 369,411-414). In addition to direct recruitment by protein-protein bindinginteractions, protein-lipid binding interactions are also important fortranslocation (Nishizuka, Y. (1992) Science 258, 607-614; Rameh, L. E. &Cantley, L. C. (1999) J. Biol. Chem. 274, 8347-8350). Lipid recruitmentis exemplified by the translocation of PH-domain containing proteins inresponse to receptor-mediated production of plasma membranephosphatidylinositol lipids (Ferguson, et al. (1995) Cell 83,1037-1046), C1-domain containing proteins in response to plasma membranediacylglycerol production, and C2-domain containing proteins in responseto calcium mediated binding interactions with negatively charged lipidsin the plasma membrane (Nishizuka, Y. (1992) Science 258, 607-614;Newton, A. C. (1995) Curr. Biol. 5, 973-976).

Why does translocation to the plasma membrane play such a ubiquitousrole in signal transduction? First, most of the cellular interactionswith the extracellular environment are mediated by receptors located inthe plasma membrane. Activated receptors often serve as a scaffold forsignaling proteins that have to be recruited for a particular signalingfunction. Second, plasma membrane translocation concentrates signalingproteins at the membrane and enhances the frequency of intermolecularcollisions. Translocation then serves as an intermediate signaling stepthat enhances the effective on-rate for target binding or the Michaelisconstant for enzyme action (Haugh, J. M. & Lauffenberger, D. A. (1997)Biophys. J. 72, 2014-2031).

Over the last few years, confocal imaging measurements were used tomonitor the plasma membrane translocation of signaling proteins overtime (Sakai, et al. (1997) J. Cell Biol. 139, 1465-1476; Venkateswarlu,et al. (1998) Curr. Biol. 8, 463-466; Barak, et al. (1997) J. Biol.Chem. 272, 27497-27500; Oancea, et al. (1997) J. Cell Biol. 140,485-498; Stauffer, T. & Meyer, T. (1997) J. Cell Biol. 139, 1447-1454;Stauffer, et al. (1998) Curr. Biol. 8, 343-346; Kontos, et al. (1998)Mol. Cell Biol. 18: 4131-4140; Oancea, E. & Meyer, T. (1998) Cell 95,307-318; Parent, et al. (1998) Cell 95, 81-91; Meili, et al. (1999) EMBOJ. 18, 2092-2105; Watton, S. J. & Downward, J. (1999) Curr. Biol. 9,433-436). Although successful for many proteins, this approach waslimited to cell types where the confocal resolution was sufficient toseparate the plasma membrane from the cytosol and where thetranslocation involved a significant fraction of the cytosolic protein.Nevertheless, these imaging studies showed that single cell time-coursemeasurements of translocation events can give important insights intothe activation mechanism of enzymes (Oancea, E. & Meyer, T. (1998) Cell95, 307-318), into spatial gradients of second messengers (Parent, etal. (1998) Cell 95, 81-91; Meili, et al. (1999) EMBO J. 18, 2092-2105;Watton, S. J. & Downward, J. (1999) Curr. Biol. 9, 433-436) and into thesingle cell kinetics of specific signaling steps (Stauffer, T. & Meyer,T. (1997) J. Cell Biol. 139, 1447-1454; Oancea, E. & Meyer, T. (1998)Cell 95, 307-318).

Biomolecular or combinatorial arrays have provided a means for the highthroughput screening of chemical libraries. See, e.g., U.S. Pat. No.5,143,854. A variety of specific techniques for carrying out theautomated screening of such arrays have been developed, including theevanescent scanning of a pixel array. See U.S. Pat. No. 5,633,724.

A disadvantage of combinatorial arrays is that they provide an in vitrorather than an in vivo assay. In vitro binding assays can seldom providean accurate measure of how binding will actually occur in vivo,particularly for intracellular binding events, because the complexity ofthe intracellular environment is difficult to replicate outside of thecell. Of course, the ultimate application of many screening assays is todevelop in vivo applications for the compounds being screened.Accordingly, there is a continued need for new in vivo screeningtechniques that can be readily adapted to automated or high throughputscreening.

SUMMARY OF THE INVENTION

A first aspect of the present invention is an apparatus for screeningfor translocation of a first protein of interest in vivo in a cell. Theapparatus comprises:

(a) a total internal reflection member having a surface portion. Ifdesired, the surface portion can be divided into separate and discretesegments.

(b) A cell contacted to the surface portion by the plasma membrane ofthe cell, the protein having a fluorescent group conjugated thereto. Ifdesired, different cells can be contacted to different ones of theseparate and discrete segments.

(c) A light source operatively associated with the total internalreflection member and positioned for directing a source light into themember to produce an evanescent field adjacent the surface portion, withthe evanescent field extending into a first portion of the cell adjacentthe plasma membrane, with the evanescent field being weaker in a secondportion of the cell, the fluorescent group emitting light when in thefirst portion of the cell and emitting less light when in the secondportion of the cell (i.e., less light as compared to the amount emittedwhen the same fluorescent group is in the first portion of the cell).

(d) A light detector operatively associated with the total internalreflection member and configured to detect emitted light from the cell

The emission of more or less light from the cell indicates thetranslocation of the first protein between the first and second portionsof the cell.

The cell or cells may further contain a second protein of interestlocated in either the first portion of the cell or the second portion ofthe cell, whereby the emission of more or less light from the cellindicates the presence or absence of specific binding between the firstand second proteins of interest. When the second protein is located inthe first portion of the cell, the emission of more light indicates thespecific binding of the proteins of interest, and the emission of lesslight indicates the lack of such binding. When the second protein islocated in the second portion of the cell, the emission of less lightindicates the specific binding of the proteins of interest, and theemission of more light indicates the lack of such binding. First andsecond proteins of interest may be members of a specific binding pair.Either or both of the first and second proteins of interest may beexpressed by a nucleic acid carried by the cell; either of the first andsecond proteins of interest may be a member of a library of compounds,with a different member of said library being expressed in cells ofdifferent segments, while the other protein of interest is the same inthe cells of different segments, to provide a way to rapidly screen thelibrary of compounds.

A second aspect of the present invention is a method of detectingtranslocation of a first protein of interest within a cell. The methodcomprises:

(a) providing a total internal reflection member having a surfaceportion, with a cell contacted to the surface portion by the plasmamembrane of the cell;

(b) directing a source light into the member to produce an evanescentfield adjacent the surface portion, with the evanescent field extendinginto a first portion of the cell adjacent the plasma membrane, theevanescent field being weaker in a second portion of the cell; whereinthe protein of interest has a fluorescent group conjugated thereto; thefluorescent group emitting light when in the first portion of the celland emitting less light when in the second portion of the cell; and then

(c) detecting emitted light from the fluorescent group, with theemission of more or less light from the fluorescent group indicating thetranslocation of the first protein of interest between the first andsecond portions of the cell.

The method may be used with a second protein of interest as described inconnection with the apparatus above. An analysis of a test compound(e.g., a member of a library of compounds as described below) may becarried out by administering a test compound to the cell to determinewhether or not said test compound disrupts the binding of said first andsecond proteins of interest. The analysis may be made a quantitativeanalysis by repeating steps (a) through (c) with different cells atdifferent concentrations of said test compound. The degree of binding ordisruption of binding may then be determined at different concentrationsof the test compound.

The methods and apparatus of the invention can be used on individualcells or for screening multiple cell populations, or libraries of cellsor libraries of compounds, as described in greater detail below.

A further aspect of the present invention is a method of screeningbinding between a first protein of interest and a library of secondproteins of interest within a plurality of cells. The method comprises:

(a) providing a total internal reflection member having a surfaceportion, the surface portion having a plurality of separate and discretesegments, with a cell contacted to each of the surface portion segmentsby the plasma membrane of the cells;

(b) directing a source light into the member to produce an evanescentfield adjacent the surface portion, with the evanescent field extendinginto a first portion of the cell adjacent the plasma membrane, theevanescent field being weaker in a second portion of the cell; whereinone of the proteins of interest has a fluorescent group conjugatedthereto, and the other of the proteins of interest is located in eitherthe first portion of the cell or the second portion of the cell; andwherein one of the proteins of interest is the same in each of thecells; and the other of the proteins of interest is a different memberof the library in cells contacted to different segments; with thefluorescent group emitting light when in the first portion of each ofthe cells and emitting less light when in the second portion of each ofthe cells; and then

(c) detecting emitted light from each of the segments, with the presenceor absence of emitted light indicating the presence or absence ofspecific binding between the proteins of interest in the cell in each ofthe segments.

A further aspect of the present invention is a method of screening alibrary of compounds for the ability to disrupt binding between firstand second proteins of interest. The method comprises:

(a) providing a total internal reflection member having a surfaceportion, the surface portion having a plurality of separate and discretesegments, with a cell contacted to each of the surface portion segmentsby the plasma membrane thereof;

(b) directing a source light into the member to produce an evanescentfield adjacent the surface portion, with the evanescent field extendinginto a first portion of the cell adjacent the plasma membrane, theevanescent field being weaker in a second portion of the cell;

wherein one of the proteins of interest has a fluorescent groupconjugated thereto, and the other of the proteins of interest is locatedin either the first portion of the cell or the second portion of thecell;

the fluorescent group emitting light when in the first portion of eachof the cells and emitting less light when in the second portion of eachof the cells;

 then

(c) administering a different member of the library of compounds to eachof the separate and discrete segments (e.g., by contacting a differentcompound to the cells, or by expressing a different compound from adifferent nucleic acid in each of said cells); and then

(d) detecting emitted light from the fluorescent group in the cells fromeach of the separate and discrete segment.

The presence or absence of emitted light from the fluorescent groupindicates the disruption or lack of disruption of specific bindingbetween the proteins of interest by the member of the libraryadministered to the segment.

When screening libraries, the screening steps may be repeated withdifferent members of the library until sufficient members of the libraryhave been screened. It will also be appreciated that each cell maycontain or be administered a sub-population or subpool of the library,and that where a population or subpopulation is found to contain acompound having desired properties, the screening step may be repeatedwith additional subpopulations containing the desired compound until thepopulation has been reduced to one or a sufficiently small number topermit identification of the compound desired.

A further aspect of the present invention is an apparatus for screeningfor translocation of a first protein of interest in vivo in a cell. Theapparatus comprises:

(a) a thin unitary total internal reflection member having a surfaceportion. If desired, the surface portion can be divided into separateand discrete segments. The total internal reflection member ispreferably a single thin member which can be conveniently formed from amicroscope slide coverslip, although other embodiments are alsocontemplated.

(b) A cell (typically a plurality of cells) contacted to the surfaceportion by the plasma membrane of the cell, the protein having afluorescent group conjugated thereto. If desired, different cells can becontacted to different ones of the separate and discrete segments.

(c) A light source operatively associated with the total internalreflection member and positioned for directing a source light into themember to produce an evanescent field adjacent the surface portion, withthe evanescent field extending into a first portion of the cell adjacentthe plasma membrane, with the evanescent field being weaker in a secondportion of the cell, the fluorescent group emitting light when in thefirst portion of the cell and emitting less light when in the secondportion of the cell (i.e., less light as compared to the amount emittedwhen the same fluorescent group is in the first portion of the cell).

(d) coupling means such as a cylindrical lens or lenses, or a focusedlaser used in combination with a one-dimensional scanning mirror, etc.,for coupling the light source to the thin unitary total internalreflection member, preferably thereby providing wide-field illuminationof the surface portion of the total internal reflection member.

(e) A light detector operatively associated with the total internalreflection member and configured to detect emitted light from the cell

The emission of more or less light from the cell indicates thetranslocation of the first protein between the first and second portionsof the cell.

The cell or cells may further contain a second protein of interestlocated in either the first portion of the cell or the second portion ofthe cell, whereby the emission of more or less light from the cellindicates the presence or absence of specific binding between the firstand second proteins of interest. When the second protein is located inthe first portion of the cell, the emission of more light indicates thespecific binding of the proteins of interest, and the emission of lesslight indicates the lack of such binding. When the second protein islocated in the second portion of the cell, the emission of less lightindicates the specific binding of the proteins of interest, and theemission of more light indicates the lack of such binding. First andsecond proteins of interest may be members of a specific binding pair.Either or both of the first and second proteins of interest may beexpressed by a nucleic acid carried by the cell; either of the first andsecond proteins of interest may be a member of a library of compounds,with a different member of said library being expressed in cells ofdifferent segments, while the other protein of interest is the same inthe cells of different segments, to provide a way to rapidly screen thelibrary of compounds.

A further aspect of the present invention is a method of detectingtranslocation of a first protein of interest within a cell. The methodcomprises:

(a) providing a thin unitary total internal reflection member having asurface portion, with a cell contacted to the surface portion by theplasma membrane of the cell;

(b) directing a source light into the member through a coupling means toproduce a wide-field evanescent field adjacent the surface portion, withthe evanescent field extending into a first portion of the cell adjacentthe plasma membrane, the evanescent field being weaker in a secondportion of the cell; wherein the protein of interest has a fluorescentgroup conjugated thereto; the fluorescent group emitting light when inthe first portion of the cell and emitting less light when in the secondportion of the cell; and then

(c) detecting emitted light from the fluorescent group, with theemission of more or less light from the fluorescent group indicating thetranslocation of the first protein of interest between the first andsecond portions of the cell.

The method may be used with a second protein of interest as described inconnection with the apparatus above. An analysis of a test compound(e.g., a member of a library of compounds as described below) may becarried out by administering a test compound to the cell to determinewhether or not said test compound disrupts the binding of said first andsecond proteins of interest. The analysis may be made a quantitativeanalysis by repeating steps (a) through (c) with different cells atdifferent concentrations of said test compound. The degree of binding ordisruption of binding may then be determined at different concentrationsof the test compound.

The methods and apparatus of the invention can be used on individualcells or for screening multiple cell populations, or libraries of cellsor libraries of compounds, as described in greater detail below.

The present invention is explained in greater detail in the drawingsherein and the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of an evanescent microscope of theinvention, useful for carrying out a quantitative translocation analysis(QTA).

FIG. 2 demonstrates a quantitative translocation analysis carried outwith an apparatus of FIG. 1.

FIG. 3 demonstrates a photomultiplier simulation of a quantitativetranslocation analysis signal.

FIG. 4 shows PAF induced plasma membrane translocation of C1-GFPmeasured by evanescent microscopy with an apparatus of FIG. 1.

FIG. 5 illustrates an apparatus of the present invention for screening aplurality of cells.

FIG. 6. Evanescent wave excitation of fluorescent cytosolic and membraneproteins in adherent cells. (A) Schematic view of the microscope setup(see Materials and Methods section for description). (B) Fluorescentimages of RBL cells that express cytosolic GFP (left; representative of6 experiments) or plasma membrane targeted GFP (right, n=3). Amyristoylation/palmitoylation sequence was fused to the N-terminal endof GFP for membrane targeting. While expressed at a similar level, themembrane targeted GFP exhibited a 3 to 10-fold higher fluorescenceintensity than the cytosolic GFP (Bar=10 μm).

FIG. 7. Plasma membrane translocation of PKCγ measured in hippocampalastrocytes by evanescent wave excitation. (A) Time series of fluorescentimages of cultured hippocampal astrocytes expressing PKCγ-GFP stimulatedwith glutamate (100 μM; n=47). A marked increase in fluorescenceintensity was observed. (B) The same experiment as in (A) but withcalcium ionophore added (2 μM ionomycin; n=8). (Bars=10 μm in (A) and(B)). (C) Time course of PKCγ-GFP translocation measured as a fractionalincrease in fluorescence intensity and triggered by glutamate addition(100 μM, left) to a single astrocyte (left panel; n=47). (D) Similarperiodic intensity changes in an RBL stably expressing PAF receptorsfollowing the addition of 100 nM PAF. Ionomycin was added at the end ofeach experiment (2 μM).

FIG. 8. Measurement of the plasma membrane translocation of GFP-taggedminimal protein domains. (A) Translocation of GFP-PH domain of Akt (oralso termed PKB) triggered by addition of PAF (1 μM) to RBL cells (n=2).Cells were kept for 10 hours in serum-free medium before stimulation.(B) Translocation of C1₂-GFP domain from PKCδ triggered by addition ofdiacylglycerol (DiC8, 0.5 mM) to cultured hippocampal astrocytes (n=12).(Bars=10 μm).

FIG. 9. Local translocation and cell-to-cell variability oftranslocation events. (A) Stimulation of astrocytes with lowconcentrations of glutamate triggered local transient translocationevents of expressed GFP-C2 (PKCγ) domain (n=2). Since this C2 domain hasbeen shown to bind negatively charged lipids in the presence of calcium,these local translocation events likely reflect local increases incalcium concentration. Bar=10 μm. (B and C) Statistical analysis ofGFP-C2 domain translocation in PAF-receptor expressing RBL-cellsstimulated with PAF (100 nM). (B) Typical time course of thestimulus-induced repetitive translocation events in an individual cell.(C) Analysis of the relative fluorescent increase of C2 domaintranslocation of 16 cells in the field of view. The histogram shows thenumber of cells with a given relative fluorescence increase,R=(I₁−I₀)/(I₀−BG), where I₀ represents the basal fluorescence intensityand I₁ is the intensity after the maximal translocation of thefluorescent construct. BG is the background value.

FIG. 10. Measuring protein-protein binding interactions of cytosolicproteins using an evanescent wave excitation. (A) Schematicrepresentation of the in vivo binding assay. Binding between Proteins Xand Y can be measured by conjugating Protein X with GFP and Protein Ywith an inducible plasma membrane targeting domain. (B) Demonstrationthat evanescent wave excitation can be used to measure bindinginteractions between two CaMKII isoforms. The C1A-domain from PKCγ wasused as an inducible translocation domain. (Left) Phorbol ester addition(PDBu, 1 μM) to RBL-cells that express C1A-CaMKIIα and GFP-CaMKIIαtriggered a marked increase in plasma membrane fluorescence, consistentwith the previous findings that CaMKIIα forms multimers (n=3). (Right)Phorbol ester addition to RBL-cells that express C1A-CaMKIIβ andGFP-CAMKIIα lead to a similar fluorescence increase, demonstrating thatCaMKIIα and CaMKIIβ bind to each other in the cytosol (n=2). Typicaltranslocation traces are shown.

FIG. 11. E-SCAM measurement of plasma membrane translocation events inmore than a thousand cells. (A) Schematic view of the Evanescent waveSingle Cell Array Macroscope (E-SCAM) in which the excitation light isdirectly coupled into the coverslip. (B) Low magnification images ofRBL, cells expressing PKCγ-YFP before (left) and after stimulation withcalcium ionophore (right). The insert shows a more detailed view of asubregion of the image. (Bar=1 mm). (C and D) Statistical analysis ofreceptor-triggered fluorescence intensity changes measured in RBL-cellsexpressing PKCγ-YFP after stimulation by cross-linking of their FcεRIreceptors with the antigen DNP-BSA. (C) Typical time courses. (D)Histogram of the maximum amplitude of fluorescence intensity changesthat was measured in each of more than 1000 cells visible in the fieldof view.

FIGS. 12 and 13 illustrate schematic diagrams representative of theapparatuses of the invention. Although specific embodiments are setforth therein, it should be appreciated that modifications to theseembodiments can be made without altering the scope of the invention. Forexample, lenses of different shapes, dimensions, and geometries can beemployed, as well as different lasers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

The term “contact” or contacted to”, when used herein with respect to acell or cells contacted to a total internal reflection member, meanssufficiently close to be excitable by the evanescent wave. This meansthat, in a typical configuration, the total internal reflection memberis less than 5 micrometer away from the cell membrane. Such contact maybe direct or indirect through intervening materials (e.g, cell adhesionproteins or extracellular matrix).

Total internal reflection (TIR) members useful for carrying out thepresent invention include prisms, waveguides, fibers, and specializedmicroscope objectives. The member may be a single unitary element or acombination of elements (for example, a glass slide contacted to a prismby an intervening oil). In the case of waveguides and fibers, there maybe several TIR surfaces. Additionally, the TIR surface can be on anoptically transparent substrate surface such as a glass slide, opticalfilm, or the like, that is optically coupled with the TIR element in aconventional manner, for example, using a refractive index matching oilor a compressible optical polymer such as those disclosed by Sjodin,“Optical interface means,” PCT publication WO 90/05317, 1990. Thesubstrate surface is preferably removable from the TIR element, and mayeven be disposable. If the test cell or cells are contacted directly tothe TIR element, such as the prism, it may be necessary to clean orreplace the prism before testing other cells, and the alignment of theprism will then have to be checked and possibly readjusted. Providing aremovable substrate (e.g. by means of refractive index matching oil on,for example, a prism) eliminates or at least greatly reduces the costand effort involved in ensuring that the prism is clean and aligned.

The TIR members may be present in a variety of shapes and geometries. Inone embodiment of the invention, the member may be a single unitaryelement or a combination of elements (for example, a glass slidecontacted to a prism by an intervening oil), but in the instantinvention is a preferably a single thin unitary element. In the case ofwaveguides and fibers, there may be several TIR surfaces. If the testcell or cells are contacted directly to the TIR element, such as theprism, it may be necessary to clean or replace the prism before testingother cells, and the alignment of the prism will then have to be checkedand possibly readjusted. Providing a removable substrate (e.g. by meansof refractive index matching oil on, for example, a prism) eliminates orat least greatly reduces the cost and effort involved in ensuring thatthe prism is clean and aligned.

The TIR member may have various dimensions that can be selected by theskilled artisan. Preferably, the thickness of the member range fromabout 100 to about 5000 micrometers. The width of the member preferablyranges from about 5 or 6 mm to about 300 mm, and the depth of the memberpreferably ranges from about 5 or 6 mm to about 300 mm. A preferredmember is 200 micrometers thick, 22 mm wide and 22 mm deep.

An advantage of coupling the light source directly into a thin unitaryTIR element such as a coverslip is the much improved geometry forimaging, and in particular directly into the side or edge of the thinunitary TIR element (e.g., glassplate or coverslip). The excitationpathway is now essentially out of the way from the emission pathway sothat an inverted wide-field microscope can be used. Furthermore, sincethe laser light is reflected several times from the illuminated areaduring its passage into the coverslip, significantly less laser power isneeded to obtain the same overall brightness of illumination.

By “wide field” is meant that an area of the surface portion of thetotal internal reflection member of at least 10, 15, 20 or 30 squaremillimeters is effectively illuminated by the light source. Such an areacan contain 6, 10, 20 or 30 or more separate and discrete regions, allof which may contain separate and discrete cell populations, e.g., spotson a thin unitary TIR element or wells in a multi-well cell chamber, aswell as others.

Light sources suitable for carrying out the present invention include,but are not limited to, lasers, LEDs, coherent frequency-convertingdevices (an example of which is disclosed by Kozlovsky et at.,“Resonator-enhanced frequency doubling in an extended-cavity diodelaser,” presented at Blue/Green Compact Lasers, New Orleans Feb. 1-5,1993 and references therein), an array of surface emitting LEDs (Bare etal., “A simple surface-emitting LED array useful for developingfree-space optical interconnects,” IEEE, Photon. Tech. Lett., Vol. 5,172-175, 1993), and a suitable array of vertical-cavity surface-emittinglasers (VCSEL) where each polymer array pixel could have its owncorresponding laser on the VCSEL array (Salah and Teich, Fundamentals ofPhotonics, Wiley-Interscience, New York, 1991, p. 638). Examples ofmolecular tag/light source pairs include CY5/HeNe laser, CY5/laser diode(e.g. Toshiba TOLD9410(s)), CY5/LED (e.g. Hewlett-Packard HMP8150),fluorescein/argon ion laser, and rhodamine/argon ion laser.

Any suitable optical coupling means can be used to carry out the presentinvention, including but not limited to cylindrical lenses, a focussedlaser scanned into the TIR by a one-dimensional scanning mirror, etc.

Any suitable light detector may be used to carry out the presentinvention. An example of a suitable detection system is shown in U.S.Pat. No. 5,633,724 to King et al. As illustrated therein, an imagingsystem collects and images the optical signal through an optical filterand onto a two-dimensional array detector. Imaging systems can containlenses or a coherent fiber bundle. The filter is chosen to transmit theoptical signal and reject radiation at other frequencies. The detectoris preferably a two dimensional detector such as CCD array, imageintensified CCD, vidicon or video camera. An optional image intensifier,such as a Hamamatsu V4170U image intensifier, can be used in addition todetector if the optical signal is weak.

Cells used to carry out the present invention are typically eukaryoticcells, which may be yeast, plant, or animal cells. Yeast and animalcells, particularly mammalian cells, are currently preferred. Exampleplant cells include, but are not limited to, arabidopsis, tobacco,tomato and potato plant cells. Example animal cells include, but are notlimited to, human, monkey, chimpanzee, rat, cat, dog, and mouse cells.In one embodiment, each of the cells is adherent to the thin unitary TIRelement. Many of the cells are present within a spot (e.g., subregion)or a well in the instance when barriers are used between thepopulations. For such an embodiment to work properly, it is preferredthat the cells adhere directly to the thin unitary TIR element or acoating thereon (e.g., less that about 500 nm away from the surface).

“Detectable groups” or “detectable proteins” used to carry out thepresent invention include fluorescent proteins, such as greenfluorescent protein (GFP) and apoaequorin, including analogs andderivatives thereof. Green fluorescent protein is obtained from thejellyfish Aequorea victoria and has been expressed in a wide variety ofmicrobial, plant, insect and mammalian cells. A. Crameri et al., NatureBiotech. 14, 315-319 (1996). Any detectable group may be employed, andother suitable detectable groups include other fluorophores orfluorescent indicators, such as a fusion tag with any binding domainsuch as avidin, streptavidin and ligand binding domains of receptors.Coupling of biotin or other ligands to the fluorophore or indicator ofinterest may be achieved using a dextran matrix or other linker system.The detectable protein may be one which specifically binds afluorophore, as in FLASH technology. Fluorescent detectable groups(including both fluorescent proteins and proteins that bind a separatefluorophore molecule thereto) are currently preferred.

“Internal structure” as used herein refers to a separate, discreet,identifiable component contained within a cell. The term “structure” asapplied to the constituent parts of a cell is known (see, e.g., R.Dyson, Cell Biology: A Molecular Approach, pg, 10 (2d ed. 1978)), andthe term “internal structure” is intended to exclude external structuressuch as flagella and pili. Such internal structures are, in general,anatomical structures of the cell in which they are contained. Examplesof internal structures include both structure located in the cytosol orcytoplasm outside of the nucleus (also called “cytoplasmic structures”),and structures located within the nucleus (also called “nuclearstructures”). The nucleus itself including the nuclear membrane areinternal structures. Structures located within the cytoplasm outside ofthe nucleus are currently preferred. Thus the term “internal structure”is specifically intended to include any non-uniformly distributedcellular component, including proteins, lipids, carbohydrates, nucleicacids, etc., and derivatives thereof.

“Library” as used herein refers to a collection of different compounds,typically organic compounds, assembled or gathered together in a formthat they can be used together, either simultaneously or serially. Thecompounds may be small organic compounds or biopolymers, includingproteins and peptides. The compounds may be encoded and produced bynucleic acids as intermediates, with the collection of nucleic acidsalso being referred to as a library. Where a nucleic acid library isused, it may be a random or partially random library, commonly known asa “combinatorial library” or “combinatorial chemistry library”, or itmay be a library obtained from a particular cell or organism, such as agenomic library or a cDNA library. Small organic molecules can beproduced by combinatorial chemistry techniques as well. Thus in general,such libraries comprise are organic compounds, including but not limitedoligomers, non-oligomers, or combinations thereof. Non-oligomers includea wide variety of organic molecules, such as heterocyclics, aromatics,alicyclics, aliphatics and combinations thereof, comprising steroids,antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids,opioids, benzodiazepenes, terpenes, prophyrins, toxins, catalysts, aswell as combinations thereof. Oligomers include peptides (that is,oligopeptides) and proteins, oligonucleotides (the term oligonucleotidealso referred to simply as “nucleotide, herein) such as DNA and RNA,oligosaccharides, polylipids, polyesters, polyamides, polyurethanes,polyureas, polyethers, poly (phosphorus derivatives) such as phosphates,phosphonates, phosphoramides, phosphonamides, phosphites,phosphinamides, etc., poly (sulfur derivatives) such as sulfones,sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for thephosphorous and sulfur derivatives the indicated heteroatom for the mostpart will be bonded to C, H, N, O or S, and combinations thereof. See,e.g, U.S. Pat. No. 5,565,324 to Still et al., U.S. Pat. No. 5,284,514 toEllman et al., U.S. Pat. No. 5,445,934 to Fodor et al. (the disclosuresof all United States patents cited herein are to be incorporated hereinby reference in their entirety).

“Nucleic acid” as used herein refers to both DNA and RNA.

“Protein” as used herein is intended to include protein fragments, orpeptides. Thus the term “protein” is used synonymously with the phrase“protein or fragment thereof” (for the purpose of brevity), particularlywith reference to proteins that are “proteins of interest” or members ofa specific binding pair. Protein fragments may or may not assume asecondary or tertiary structure. Protein fragments may be of any length,from 2, 3, 5 or 10 peptides in length up to 50, 100, or 200 peptides inlength or more, up to the full length of the corresponding protein.

“Specifically binds” and “specific binding” as used herein includes butis not limited to stereospecific binding, electrostatic binding, orhydrophilic binding interactions. Thus, specifically binds and specificbinding are exhibited by at least a two or three fold (or two or threetimes), greater apparent binding affinity between the binding partnersas compared to other proteins or binding partners within the cell inwhich binding is being detected.

“Specific binding pair” refers to a pair of molecules (e.g., a pair ofproteins) that specifically bind to one another. A pair of moleculesthat specifically bind to one another, which may be the same ordifferent, are referred to as members of a specific binding pair. Aprotein that is a member of a specific binding pair may be a proteinthat has been previously determined to be a member of a specific bindingpair or a protein that is a putative member of a specific binding pair.Examples of the latter include members of a library, such as theproducts of a cDNA or combinatorial library, or a protein for which abinding partner has not yet been identified, where it is desired toidentify a naturally occurring (e.g., a product of a cDNA or genomic DNAlibrary) or non-naturally occurring (e.g., combinatorial) bindingpartner therefore.

“Translocation” as used herein refers to a change in distribution of aprotein or conjugate (including a fusion protein) from one physicaldistribution within a cell to another, different, physical distributionwithin a cell. Preferably, translocation is from either a uniform ornon-uniform distribution to a non-uniform distribution. Translocationcould also be from a non-uniform to a uniform distribution.Translocation may be induced by any suitable means, such as byadministration of a physical or chemical signal (e.g., administration ofa compound such as a phorbol ester or platelet activating factor (PAF)).Many signal transduction proteins are known to change their distributionafter stimulation of the corresponding receptor (or other appropriatestimulus), and can be used to carry out the present invention. Oftenthese translocation events are mediated by subdomains of such signalingproteins (e.g., the C1 or C2 subdomains), and such subdomains can beused to carry out the present invention.

As noted above, the present invention provides a method of detecting aprotein-protein interaction. The method comprises first providing a cellthat contains a first heterologous conjugate and a second heterologousconjugate. The first heterologous conjugate comprises a first protein ofinterest conjugated to a detectable group. The second heterologousconjugate comprises a second protein of interest (which may be the sameas or different from the first protein of interest) conjugated to aprotein that specifically binds to an internal structure within thecell. The binding of the protein that specifically binds to an internalstructure may be immediate, may be induced (as discussed below), or maybe a prior binding in the case of a protein that is previously localizedto or permanently located at the internal structure of interest. The twoconjugates are preferably each present in the cell at a totalconcentration between about 1 or 10 nM to about 1 or 10 mM.

The presence or absence of binding of the detectable group to theinternal structure is then detected, the presence of the bindingindicating that the first and second proteins of interest specificallybind to one another. Detection may be by any suitable means dependingupon the detectable group employed, but preferably the detectable groupis a fluorescent group and detection is carried out by optical or visualreading, which may be done manually, by an automated apparatus, or bycombinations thereof.

If desired, the second heterologous conjugate can further comprise adetectable group, which detectable group is preferably different fromthe detectable group located on the first heterologous conjugate andfluoresces at a different wavelength therefrom. For example, bothdetectable groups could be a green fluorescent proteins, yet simplydifferent mutants of green fluorescent protein that fluoresce atdifferent wavelengths.

Either or both of the heterologous conjugates may be introduced directlyin the cell by any suitable means, such as by electroporation orlipofection. In the alternative, when the heterologous conjugates arefusion proteins, a nucleic acid (typically a DNA) may be stableintroduced into the cell (for example, as a plasmid), which nucleic acidincludes a suitable promoter segment that controls and causes theexpression of a nucleic acid encoding the fusion protein. Again, eitheror both of the fusion proteins may be produced in the cell in thismatter.

Binding events in the instant invention may be direct or indirectbinding events. Indirect binding events are those mediated through anintermediate, or bridging, molecule or conjugate. Administration of sucha bridge molecule can be a signal to induce translocation (discussedbelow). For example, the bridging molecule may be a covalent conjugateof FK506 and cyclosporin, to cause the indirect binding of FKBP12 andcyclophilin (both conventionally cytosolic proteins) to one another.Either of the FKBP12 or the cyclophilin can be modified so that it bindsto the plasma membrane, such as by lipidating the protein or forming afusion protein with the transmembrane domain of a transmembrane protein.

Any internal structure as defined above can be used to carry out thepresent invention, as long as the binding of the detectable group to theinternal structure provides a different detectable signal from the cellthan when the detectable group is not bound to the internal structure.In one preferred embodiment the internal structure is contained in thecell cytoplasm. Examples of internal structures include, but are notlimited to, plasma membrane, cytoskeleton (including but not limited toactin cytoskeleton, tubulin cytoskeleton, intermediate filaments, focaladhesions, etc.), centromere, nucleus, mitochondria, endoplasmicreticulum, vacuoles, golgi apparatus, and chloroplasts. Preferably, theinternal structure is either the plasma membrane or corticalcytoskeleton.

In a preferred embodiment of the invention, the protein thatspecifically binds to an internal structure is a translocatable protein.In this embodiment, the method further comprises the step of inducingtranslocation of the second heterologous conjugate prior to thedetecting step. Induction of translocation may be carried out by anysuitable means, such as by administration of a physical or chemicalsignal (e.g., administration of a compound such as a phorbol ester).Such a protein may be selected from the group consisting of cytosolicprotein kinases, protein phosphatases, adapter proteins, cytoskeletalproteins, cytoskeleton associated proteins, GTP-binding proteins, plasmatransmembrane proteins, plasma membrane associated proteins, β-arrestin,and visual arrestin (including fragments thereof that specifically bindto an internal structure). Preferably, the protein is a protein kinase Cisoform or a fragment thereof that specifically binds to an internalstructure, such as a C1 domain fragment or a C2 domain fragment ofprotein kinase C gamma (or other suitable protein kinase C), where theinduction signal is administration of a phorbol ester. In addition,induction of translocation may be induced by stimulation of a receptor,such as a glutamate receptor, beta-adrenergic receptor, or PAF receptor,with a receptor agonist to induce a signaling step which in turn inducestranslocation. Finally, numerous proteins may be modified to make themtranslocatable by employing bridging molecules, as discussed above.

As noted above, in one embodiment of the invention the first and secondproteins of interest may together comprise members of a specific bindingpair. In this embodiment, the invention may further include the step ofadministering a test compound to the cell prior to the detecting step,wherein the absence of binding of the detectable group to the internalstructure indicates that the test compound inhibits the binding of themembers of the specific binding pair. Any test compound can be used,including peptides, oligonucleotides, expressed proteins, small organicmolecules, known drugs and derivatives thereof, natural or non-naturalcompounds, etc. Administration of the test compound may be by anysuitable means, including direct administration such as byelectroporation or lipofection if the compound is not otherwise membranepermeable, or (where the test compound is a protein), by introducing aheterologous nucleic acid that encodes and expresses the test compoundinto the cell. Such methods are useful for screening libraries ofcompounds for new compounds which disrupt the binding of a known bindingpair.

FIG. 1 is a schematic illustration of an evanescent microscope 10 of theinvention, useful for carrying out a quantitative translocation analysis(QTA). A prism 11 is used as the total internal reflection (TIR) member.The prism is a dove prism made of crown glass (part number 01PDE005,Melles Griot, Irvine, Calif.). Excitation light 12 (argon ion laser at488 nm) for generating the evanescent field is provided by an Enterpriseargon-ion laser 13 obtained from Coherent Inc., Moutainview, Calif. Aglass cover slip 14 made of crown glass has cells adhered thereto, andis contacted to the dove prism with an intervening coating of oil(Immersionsol N518 from Zeiss, Inc.). The angle of the laser light intothe TIR member is adjusted to provide an evanescent field in first and(weakly) second portions of the cells, as explained above. A ZeissAxioskop microscope stand is used as a stand for a Princeton InstrumentCCD 1300-Y 15 (Trenton, N.J.) photon detection system, which is in turnconnected to a personal computer programmed with appropriate datacollection and analysis software.

FIG. 2 demonstrates a quantitative translocation analysis carried outwith an apparatus of FIG. 1. RBL cells expressing C2-GFP were stimulatedwith PAF. The fluorescence signal average 20 was measured from 6 cells.Each cell had relative signal increases that ranged from 3 to 6 fold.

FIG. 3 demonstrates a photomultiplier simulation of a quantitativetranslocation analysis signal, with the same data analyzed as for thesingle cells in FIG. 2 above. The average relative fluorescence increase21 for the entire field of view (simulating what a photomultiplier wouldencounter) is illustrated. The field of view was approximately 0.15 mmin diameter.

FIG. 4 illustrates PAF induced plasma membrane translocation of C1-GFPmeasured by evanescent microscopy with an apparatus of FIG. 1. Therelative increase in fluorescence intensity 22 for an average of sixcells is shown at 3 second time-points.

FIG. 5 illustrates an apparatus of the present invention forsimultaneously screening a plurality of cells. The apparatus comprises alight source 31, a prism 32, a substrate 33 positioned on top of theprism and optically contacted to the prism by means of an oil 34 with asimilar index of refraction to the substrate and the prism, an opticaldetection system 36 and a computer 37. The light source, prism, andsubstrate may be the same as the light source, prism and substratedescribed in Example 1 above. The substrate is divided into segments bya rigid polymer grid 40 that is secured to the substrate by means of anadhesive. Cells 45 are adhered to the substrate in each of the discreteregions, as described above. Excitation light 46 produces an evanescentfield in first regions of the cells which in turn generates a lightsignal 47 from the cells that is detectable or distinguishable bydetection system 36 when the fluorescent group is in the first region ofthe cells. The various components of the system can be used in likemanner to that described in U.S. Pat. No. 5,633,724 (the disclosure ofwhich is incorporated by reference herein in its entirety), except thatcells are employed in contrast to compounds. The discrete regions on thesubstrate can be formed by any suitable means, such as by adhering aphysical barrier such as a grid to the substrate, simply adhering cellsin different regions, forming wells, channels, grooves or other physicalbarriers in the substrate, etc. The apparatus of FIG. 5 can be used forthe simultaneous screening of a plurality of cells, which may be thesame or different.

In the embodiment of an apparatus shown in the FIGS. 12 and 13, acoverslip 100 (e.g., a 22 mm×22 mm coverslip) or the like serves as thinunitary total internal reflection member. The apparatus includes anexpanded laser 101 (e.g., 442 nm He Cd ; 514 nm Ar), a cylindrical lens102 (e.g., f=60 mm), a rotating diffuser 103, a collection lens 104(e.g., f=125 mm) and a focussing lens 105 (e.g., fn 125 mm). Thecoverslip has a polished edge portion 107 and a top surface portion 109.The laser light is focused onto a 20 mm long line on the diffuser, andthe line is projected onto the edge of the coverslip at an angle 109 ofabout 15 degrees to enable total internal reflection in the covership. Aplurality of cells 111 contacted to the surface portion 108 by theplasma membrane of the cells so that emission from proteins of interesttherein may be detected as described above. This device utilizes afluorescent imaging approach based on evanescent wave excitation (alsotermed total internal reflection fluorescence microscopy, Axelrod, D.(1981) J. Cell. Biol. 89, 141-145) to more quantitatively measure plasmamembrane translocation processes. Compared to confocal microscopy,evanescent wave imaging significantly increased (i) thesignal-to-background when measuring the fraction of plasma membranetranslocated protein, (ii) the spatial resolution when measuring themembrane distribution of localized proteins and (iii) the throughput oftranslocation measurements by enabling measurements of translocationevents simultaneously in more than a thousand cells. These studiessuggest that evanescent wave excitation of translocating fluorescentproteins and minimal protein domains is a powerful live cell imagingtool to measure signaling and binding events in the context of differentsignaling pathways and cell types.

EXAMPLE 1 Materials and Methods

Cell Culturing. Rat basophilic leukemia 2H3 (RBL) cells were grown andplated as described by Oancea, et al. ((1997) J. Cell Biol. 140,485-498) and Oancea, E. & Meyer, T. ((1998) Cell 95, 307-318). RBL cellswith stably transfected PAF receptors were obtained from the laboratoryof Ralph Snyderman (Duke University). Hippocampal astrocytes were usedfrom mixed neuron/glia cultures prepared as described by Teruel, et al.((1999) J. Neuroscience Methods 93:37-48). The cultures were maintainedat 37° C. in a 95% air, 5% CO₂ humidified incubator, and used 7 to 14days after plating.

Cloning of GFP-Fusion Constructs and Electroporation. The cloning of thecDNA encoding the C-terminal palmitoylation/myristoylation sequence fromLyn was described by Teruel, et al. ((1999) J. Neuroscience Methods93:37-48)) and the synthesis of in vitro transcribed RNA of CaMKIIα,C1-CaMKIIα and C1-CaMKIIβ were described by Yokoe, H. and Meyer, T.((1996) Nat. Biotechnol. 14, 1252-1256). The cDNAs for the full-lengthPKCγ and the C1₂ domain of PKCδ were cloned into the EGFP-N2 vector(Clontech). The C2 domain of PKCγ with an N-terminal EGFP was clonedinto pcDNA3 vector (Invitrogen). The PH-domain of Akt was cloned intothe EGFP-C1 vector (Clontech). The cDNA or RNA constructs weretransfected by a microporation device for adherent cells (Teruel, M. N.& Meyer, T. (1997) Biophys. J. 73, 1785-96; Teruel, et al. (1999) J.Neuroscience Methods 93:37-48).

Evanescent Wave Excitation and Fluorescence Microscopy (also termedTotal Internal Reflection Fluorescence Microscopy or TIRFM). Evanescentwave fluorescent excitation for studies of cells was introduced byAxelrod ((1981) J. Cell. Biol. 89, 141-145), and different versions ofsuch microscopes have been made since then (Lang et al. (1997) Neuron18, 857-863; Thompson, N. L. & Lagerholm, B. C. (1997) Curr. Opin.Biotechnol. 8, 58-64). In this method, the reflected light produces anexponentially decaying light field above the glass-water interface witha space constant given by the angle of the incident light source. Forglass coverslips, total internal reflection occurs for angles above 61°[θ_(I,c)=arcsin(n₂/n₁), calculated for a refractive index n₁=1.52 forglass and n₂=1.33 for water]. For measurements of receptor-triggeredtranslocation, we found that total internal reflection angles of 70° area suitable compromise between having a sufficiently deep penetrationdepth (which decreases with increasing angles) and generating unwantedstray light due to scattering objects in the light path (which increaseswith the angle). The space constant of the exponential decayI(z)=I(0)exp(−z/d) at a given angle can be calculated as described byAxelrod ((1981) J. Cell. Biol. 89, 141-145).

d=λ _(o)/[4π(n ₁ ²*sin²(θ_(i))−n ₂ ²))^(1/2)]=75 nm; with λ_(o)=488 nmand θ_(I)=70°  (1)

An evanescent wave microscope configuration optimized for long term livecell imaging with minimal focus drift while having free access to thebath solution for buffer exchange and cell manipulation was designed.The system was built around a Zeiss Axioskop 2 microscope with afocusable water immersion objective. The laser excitation beam enteredfrom below the coverslip through a spatially fixed dove prism (EdmundScientific). Cells were illuminated by coupling the laser into thecoverslip through a 200 μm thin microscope immersion oil layer betweenthe coverslip and the prism. Cells were grown on 25 mm square Type IIcoverslips in a chamber enclosed by a Teflon ring (3 mm wide and 3 mmhigh) that contained the extracellular buffer solution. In order to beable to look at different regions on the coverslip, the coverslipitself—but not the fixed prism and objective—was directly mounted on amotor controlled x-y stage.

The cells expressing GFP fusion proteins were excited using a 488 nmlaser line (Coherent, typically 10-100 mW) and a 500 nm long pass filterfor emission (Chroma Inc.).

A self-built rotating diffuser was used to homogenize the laser light.The diffuser is a device to make the laser light more homogeneous forthe imaging. It consists of a conventional motor that has a shaft with alight shaping diffuser mounted at the end of it (we have tried 0.5 and 1degree diffusers from Physics Optics Corporation, and both work verywell). By rapidly rotating the diffuser the laser light becomes morehomogeneous than by just leaving the laser on the same spot on thediffuser.

An 85 mm focal distance lens was used to image the scrambled lightsource onto the prism. The light emitted by the fluorescent proteins wascollected by a cooled CCD videocamera (Micromax, 5 MHz, PrincetonInstrument). Time series of images were recorded using Metamorphsoftware (Universal Imaging). Experiments were carried out at roomtemperature (˜25° C.).

Construction of an Evanescent wave Single Cell Array Macroscope(E-SCAM). A wide-field microscope (macroscope) was designed in similarfashion to the higher magnification system described above. TwoRodenstock 100 mm Heligon lenses were mounted below the coverslip inopposite directions to obtain a combined objective and ocular with 1:1magnification. The second lens projected the sample image onto a 5 MHzMicromax camera made commercially available from Roper Scientific ofTrenton, N.J. This lens combination has high spatial and chromaticaccuracy as well as a numerical aperture of NA˜0.3 (F/1.6).

An important feature of this E-SCAM design is the coupling of thescrambled laser light directly into the polished edge of a coverslip(Type II, 22×22 mm) using a cylindrical lens. The polished angle was setat 20 degrees from vertical to generate a light guide within thecoverslip. The coverslip was mounted on a z-direction adjustable stageand a teflon ring was used for containing the solution in the cellchamber.

For coupling, we used two Rodenstock cylindrical lenses, the top lens(closer to the sample) images the cell sample to infinity. The secondlens was mounted in the opposite orientation to focus the image backonto the CCD camera. The distance between the two lenses was 120millimeters, which allowed a dichroic mirror to be inserted for analternative epifluorescence excitation of the cell sample. This spacealso contains the barrier filters for the emitted light.

An important consideration is the grease or other adhesive used to fixthe cell chamber (or the wells) to the coverslip. The grease should havea refractive index >1.38 or better >1.4 in order for the coupling towork most effectively. The angle of the incident light has to beadjusted to ensure total internal reflection within the coverslip. Wehave tested this empirically by maximizing the light exiting thecoverslip while grease or adhesive is present on a calibrationcoverslip. We are currently using a Dow Corning High Vacuum Silicongrease for attaching the buffer solution-containing cell chamber to thecoverslip.

EXAMPLE 2 Results

Selective Imaging of Fluorescent Proteins at the Plasma Membrane byEvanescent Wave Excitation. An evanescent wave microscope (also termedtotal internal reflection fluorescence microscopy or TIRFM) was built toexcite and monitor fluorescence signals from the adherent plasmamembrane of living cells by selectively illuminating an ˜75 nm deepregion above the glass surface (see Material and Methods for designfeatures). The microscope was optimized for having access to cells whileperforming long term imaging experiments without a focus drift. Aschematic representation of the microscope setup is shown in FIG. 6A.

Fluorescent images were recorded from RBL-cells that had similar levelsof expressed cytosolic or plasma membrane localized GFP constructs. Thetargeting of GFP to the plasma membrane utilized a fusion of the 12amino acid palmitoylation/myristoylation plasma membrane targetingsequence from Lyn to the N-terminus of GFP (Teruel, et al. (1999) J.Neuroscience Methods 93: 37-48). The fluorescence intensity of cellswith expressed cytosolic GFP (FIG. 6B) was typically 3 to 10-fold lowerthan the intensity of cells with expressed plasma membrane targeted GFP(FIG. 6C). When compared to confocal microscopy images of the samecells, evanescent wave imaging of membrane localized GFP showed a betterfine resolution of the organization of the adherent plasma membrane,suggesting that the much thinner section of excitation in evanescentwave imaging (75 nm instead of several hundred nm in confocal) markedlyreduced blurring.

Analysis of Receptor Triggered Translocation of PKCγ-GFP in Thin Cells.The dynamics of receptor-induced plasma membrane translocation ofPKCγ-GFP (Sakai, et al. (1997) J. Cell Biol. 139, 1465-1476; Oancea, etal. (1997) J. Cell Biol. 140, 485-498) was investigated in hippocampalastrocytes. The flat morphology of these cells defeated our earlierattempts to use a confocal microscopy approach for such measurements.Strikingly, glutamate stimulation led to a marked increase influorescence intensity that could be clearly resolved by fluorescenceimaging (FIG. 7A). Addition of the calcium ionophore ionomycin (FIG. 7B)also triggered a similar increase in fluorescence intensity, confirmingthat calcium signals are sufficient for translocation of PKCγ. As acontrol that the intensity increase was a result of translocation,ionomycin and phorbol ester were added to cells expressing the plasmamembrane localized GFP construct shown in FIG. 6B, and no significantchange in fluorescence intensity could be observed. The changes influorescence intensity could be followed in a series of several hundredimages. FIG. 7C shows that the typical glutamate-induced translocationevents in astrocytes showed rapid oscillations, similar to theoscillating calcium signals that have been observed previously (Yagodin,et al. (1994) J. Neurobiol. 25, 265-280). Similar receptor-triggeredfluorescence intensity oscillations could be observed in RBL cells (FIG.7D) and 3T3 fibroblasts (data not shown).

Minimal Protein Domains as Translocating Biosensors for LocalizedSignaling Events. PKCγ has at least three distinct plasma membranebinding interactions, which are mediated by two C1 domains and aC2-domain (14,33-35). This makes the translocation of the full-lengthPKC protein a complex signaling event that reflects both calcium anddiacylglycerol signals as well as potential PKC-adaptor interactions(Mochly-Rosen, D. (1995) Science 268, 247-251). Thus, owing to theirbetter defined molecular specificity, minimal protein domains are oftenbetter suited as biosensors to dissect the spatial and temporal dynamicsof a specific signaling process.

Evanescent wave imaging was used to measure the translocation of suchminimal GFP-conjugated protein domains. For example, FIG. 8A shows ameasurement of the PAF receptor stimulated production of 3′phosphorylated phosphatidylinositol lipids measured by the translocationof the GFP-tagged PH domain of Akt (Kontos, et al. (1998) Mol. CellBiol. 18: 4131-4140; Meili, et al. (1999) EMBO J. 18, 2092-2105; Watton,S. J. & Downward, J. (1999) Curr. Biol. 9, 433-436). In FIG. 8B, atandem C1 domain from PKCδ is used as a diacylglycerol reporter inastrocytes. In many cases, the translocation of minimal protein domainswas markedly local within the plasma membrane, a finding which was notpreviously apparent from confocal imaging studies. FIG. 9A shows aseries of evanescent wave images in which such localized translocationevents can be observed in glutamate stimulated astrocytes that expressthe GFP-tagged C2 domain from PKCγ. Localized translocation ofC2-domains can be explained by the earlier observations that cell-widecalcium signals are generated from localized calcium release events(Yagodin, et al. (1994) J. Neurobiol. 25, 265-280; Bootman, et al.(1997) Cell 91, 367-373; Home, J. H. & Meyer, T. (1997) Science 276,1690-1693; Parker, I. & Yao, Y. (1996) J. Physiol. (Lond) 491, 663-668).The finding of local translocation events suggests that local secondmessenger signals have a functional relevance in localized targetactivation.

Although there was a significant cell-to-cell variability in single cellmeasurements of different signaling events, a significant fraction ofcells typically respond to a receptor stimulus. An analysis of thetime-course of translocation of the C2-GFP in PAF stimulated RBL cells(FIG. 9B) shows that most cells responded with a relative peak increasesin plasma membrane fluorescence signals between 100% and 200% (FIG. 9C).

Measuring Protein-Protein Binding Interactions with HighSignal-to-Background by Evanescent Wave Excitation. Protein-proteinbinding interactions have been measured previously by using a dualprotein fusion strategy in which a protein Y was conjugated with aninducible plasma membrane translocation domain and a protein X with aGFP-tag (FIG. 5A, ref. 40). By inducing plasma membrane translocation ofthe non-fluorescent protein Y, the increase in plasma membranefluorescence from the fluorescent protein X becomes a measure for thefraction of protein X that is bound to protein Y (since it is carriedalong with protein Y to the plasma membrane). A main limitation of thisbinding assay was that the data had to be extracted by performing aconfocal image analysis of the plasma membrane versus the cytosolicdistribution of the GFP-tagged protein X.

Evanescent wave excitation was now used in combination with the sameassay. The C1A domain from PKCγ was employed as a phorbol esterinducible plasma membrane translocation domain and the same bindingpartners CaMKIIα and CaMKIIβ were investigated that were used in theprevious confocal imaging study. RNA transfection was used forquantitative expression of the two constructs (Teruel, et al. (1999) J.Neuroscience Methods 93: 37-48). Addition of phorbol ester, whichinduced the translocation of the C1A domain conjugated CaMKIIα,triggered a rapid fluorescence increase resulting from the plasmamembrane translocation of coexpressed GFP-tagged CaMKIIα (FIG. 10B),suggesting that CaMKIIα forms homo-oligomers. For the binding betweenthe α and β isoforms, CaMKIIβ was conjugated with the C1A domain andco-expressed with GFP-tagged CaMKIIα. Again, the phorbolester inducedincrease in the evanescent wave excited fluorescence signal demonstratesthat the two isoforms bind to each other (FIG. 10C). This shows thatcytosolic protein-protein binding interactions can be monitored byevanescent wave excitation in intact cells.

Simultaneous Measurements of Translocation Events in Thousands of CellsUsing an Evanescent Wave Macroscope. The studies above were performedwith high magnification in order to resolve spatial differences in thelocal distribution of GFP tagged proteins. This limits each experimentto the analysis of a few cells. In many cases, several days worth ofexperiments are needed to extract statistically significant informationfrom a specific cellular response. A significant advantage of evanescentwave imaging is that translocation measurements are reduced to anincrease in local fluorescence intensity and do not require subcellularimage analysis as is needed in confocal microscopy. The imagemagnification can therefore be significantly reduced so thattranslocation signals can be measured simultaneously from large numberof cells.

A light sensitive macroscope with a 1× magnification that projects thecoverslip surface onto a CCD camera was built. A new laser couplingstrategy was developed, in which a laser beam was focused by acylindrical lens directly into the polished and angled edge of acoverslip to generate a “coverslip light guide”. The laser light wasthen reflected numerous times within the coverslip, generating anevanescent light field in a large surface area that could be imagedwithout any need for a prism or oil coupling. The lens system and theCCD camera could then be mounted below the coverslip (FIG. 11A). Becauseof the large surface area that can be imaged in this system it wastermed an Evanescent wave Single Cell Array Macroscope or E-SCAM.

The consistency of cellular responses was tested by monitoring theionomycin-induced plasma membrane translocation of PKCγ. FIG. 11B showsan image of transfected RBL cells before and after ionomycin addition. Amarked increase in the fluorescence intensity can be seen for allindividual spots, each representing the fluorescence from an individualcell. The inset shows a small subregion at higher magnification. Thesame type of translocation events could also be observed when RBL cellswere stimulated by cross-linking FcεRI surface antigen receptors usingBSA-DNP (Oancea, E. & Meyer, T. (1998) Cell 95, 307-318). Thetime-courses of the plasma membrane translocation of PKCγ were thenanalyzed simultaneously for more than a thousand cells. FIG. 11C showstypical time-courses of such translocation events. Whileionomycin-induced translocation had a 100% cell compliance with adistribution of the induced fluorescence intensity increase between 100%and 200% of the baseline fluorescence signals (data not shown), thecompliance was slightly lower and the amplitude smaller for thetranslocation triggered by receptor stimuli (FIG. 11D). Together, thisshows that the evanescent wave excitation method is a suitablequantitative tool for monitoring signaling events simultaneously in alarge number of cells, obtaining statistically significant single cellsignaling data in a single experiment.

These studies show that evanescent wave excitation can be used forquantitative measurements of plasma membrane translocation events withhigh signal-to-background and with marked spatial resolution. Forpractical consideration, the typical z-resolution (vertical) inevanescent wave microscopy is between 0.05 to 0.1 μm while it is between0.4 to 1 μm for fluorescence images of GFP-constructs using confocalmicroscopy. The narrow z-resolution in evanescent wave excitationimproves the selective excitation of fluorescent proteins at the surfaceplasma membrane compared to fluorescent proteins in the cytosol, andthereby increases the signal for translocation measurements up to10-fold. Furthermore, the better z-resolution in evanescent wave imagingminimizes collection of light emitted from below and above the imageplane and thereby improves the spatial resolution in the x-y directionscompared to that of confocal microscopy. Finally, for GFP-taggedproteins that exchange between sites at the surface plasma membrane andother parts of the cell, evanescent wave imaging significantly reducesphotobleaching when compared to conventional imaging methods.

An important consideration in using signaling proteins or domainsconjugated with GFP is that the presence of the biosensor can alter someof the signaling responses of the native signal transduction machinery.For example, we found clear evidence for such an inhibition withfluorescently tagged SH2-domains (Stauffer, T. & Meyer, T. (1997) J.Cell Biol. 139, 1447-1454). Although this has not been stringentlytested, biosensors such as C1, C2 and PH domains that bind lipid secondmessengers and membranes are likely to require significantly higherexpressed concentrations to exhibit inhibitory effects. On the otherhand, GFP-labeled signaling enzymes are likely to function asupregulators of a particular signaling step when expressed at higherlevels than the native protein. By expressing catalytically inactivemutant constructs, and by measuring responses at differentconcentrations of the expressed constructs, it is useful to assess themagnitude of such perturbations in a specific cell type.

A second important consideration in evanescent wave translocationstudies is that plasma membrane signaling processes in the surfacecontact area can be different from those in the non-adherent side. Sinceevanescent wave imaging only excites fluorescently labeled proteins atthe plasma membrane near the surface, some signaling events may not beobserved. Ligand access to receptors at the bottom of the cell might bea problem in some cell systems, although many cells are expected toadhere to surfaces with sufficient space for small peptides and othercompounds to penetrate. These considerations suggest that a particulartranslocation process should also be investigated using confocal ordeconvolution imaging.

A notable practical difficulty in evanescent wave studies oftranslocation processes is the exponential profile of the excitationfield, which can lead to changes in fluorescence intensity caused bycell movement or cell spreading. Control experiments using plasmamembrane targeted GFP (FIG. 6) or dual fluorescence imaging with suchmarker proteins can be used to either identify or correct for suchproblems.

These studies suggest that evanescent wave imaging of translocationevents has several applications for measuring signal transductionprocesses as they occur in a living cell. Instead of having only afluorescent calcium readout, a number of diverse signaling processes cannow be measured. Such assays will be valuable for studies of signalingfeedback and cross-talk processes for different receptor-stimuli andcell types. It will be interesting to determine whether or not limitedsets of distinct “signaling states” can be defined in cells afterstimulation with single or multiple receptor inputs.

These studies also suggest that the E-SCAM technology will be useful formedium throughput screening applications. Instead of the relativelycomplex image analysis needed in confocal microscopy studies oftranslocation events, the translocation in evanescent wave imaging isreduced to a simple change in fluorescence intensity. Given the initialresults, it can be estimated that the E-SCAM method can be used tomonitor signaling processes in each of more than 10⁶ individual cells.The same type of approach can then also be used in combination with theinducible plasma membrane translocation scheme to measureprotein-protein interactions in a large cell number.

In summary, these studies suggest that evanescent wave excitation is apowerful tool in signal transduction for measuring plasma membranerecruitment and dissociation processes. It can be used to study theregulation of signaling gradients and the formation of localizedsignaling complexes, as well as to understand cross-talk and feedbackprocesses in more complex signaling systems and signal transductionnetworks. The use of evanescent wave excitation to measure responsesfrom a large number of individual cells suggests that screeningapplications become feasible for identifying pharmacological agents orexpressed protein constructs that enhance or reduce particular signalingevents or alter protein-protein binding interactions.

EXAMPLE 3 Screens

Screen 1: Identification of Chemical Libraries, Drug Collections,Peptides, Antisense and Other Compounds that Interfere with ParticularSignaling Events. Many signal transduction events result in thetranslocation of proteins between two cell portions of which one portionis near the plasma membrane. Any such signaling process that leads tosuch a translocation event can be screened using the evanescent wavemethod (i.e. calcium (C2-domain), diacylglycerol signals (C1-domains),tyrosine phosphorylation (SH2-domains), phosphatidylinositolpolyphosphate signals (different PH-domains), phosphorylation ofseven-transmembrane receptors (beta-arrestin). Establishment of celllines that express the respective translocatable fluorescentlyconjugated signaling protein or protein domain is recommended.

Useful also as secondary screen of compounds that were identified by invitro binding assays and which may or may not function as inhibitors inthe cellular environment.

Screen 2: Screen for Supressors or Enhancers of Known SignalingPathways. Screening cDNA libraries for expressed proteins that eithersuppress or enhance the signal transduction event monitored by theevanescent wave translocation assay.

Screen 3: Screen for Mutant Cells that Show Suppressed or EnhancedSignal Transduction Events. This application includes the screening ofrandomly mutated cells that show different signaling responses and thesubsequent identification of the mutated gene.

Screen 4: Identification of Ligands of Orphan Receptors. Many G-proteincoupled, tyrosine kinase and other receptors have unknown ligands. Alarge set of such ligands or drugs that may bind to the orphan receptorcan be identified with this method.

Using a translocatable fluorescent protein downstream of the receptorsuch as C1, C2, PH-domains or β-arrestin to monitor evanescent waveintensity changes.

Screen 5: Identification of Novel Binding Partners of a Known Protein.Binding interaction between the known protein X and a large number ofunknown proteins Y are screened. Protein X is made as a conjugate with afluorescent group or fluorescent protein while a library of the proteinsY is made that are each coupled to a protein or protein domain that cantranslocate between the two cell portions in response to addition of adrug, receptor stimuli or other procedure. A cell line is then madeusing the fluorescent protein X. The library of proteins Y withconjugated translocatable groups is then transfected into thesefluorescent cell lines using pooling of the library or randomtransfection. Binding partners are then identified that show a drug orstimulus induced change in fluorescence intensity. Fluoresceneintensities from individual cells or from segments of the evanescentwave apparatus surface are recorded. Either single cells are selectedfor analysis or segments of cells when pool assays are used.

The inverse screen is also possible with a library of proteins that areeach coupled to a fluorescently labeled protein and a single knownprotein that is conjugated to a protein or protein domain that cantranslocate between the two cell portions.

Screen 6: Identification of Compounds that Suppress or Enhance KnownProtein-Protein Binding Interactions. This screen is carried out withlibraries of compounds of various types, as described above.

The foregoing embodiments presented in the detailed specification,drawings, and examples are illustrative of the present invention, andare not to be construed as limiting thereof. Modifications may be madefrom these embodiments without departing from the scope of theinvention. The invention is defined by the following claims, withequivalents of the claims to be included therein.

What is claimed is:
 1. An apparatus for screening for translocation of afirst protein of interest in vivo in a cell, comprising: (a) a totalinternal reflection member having a surface portion, with (b) a cellcontacted to said surface portion by the plasma membrane of said cell,said cell containing said first protein of interest, said protein ofinterest having a fluorescent group conjugated thereto; (c) a lightsource operatively associated with said total internal reflection memberand positioned for directing a source light into said member to producean evanescent field adjacent said surface portion, with said evanescentfield extending into a first portion of said cell adjacent said plasmamembrane, said evanescent field being weaker in a second portion of saidcell, said fluorescent group emitting light when in said first portionof said cell and emitting less light when in said second portion of saidcell; and (d) a light detector operatively associated with said totalinternal reflection member and configured to detect emitted light fromsaid cell, whereby the emission of more or less light from said cellindicates the translocation of said protein between said first andsecond portions of said cell.
 2. An apparatus according to claim 1,wherein said light source comprises a coherent light source.
 3. Anapparatus according to claim 1, wherein said total internal reflectionmember comprises a prism.
 4. An apparatus according to claim 1, whereinsaid light detector comprises a CCD camera.
 5. An apparatus according toclaim 1 wherein said protein having said fluorescent group conjugatedthereto is a first protein of interest, said cell further containing asecond protein of interest located in either said first portion of saidcell or said second portion of said cell, whereby the emission of moreor less light from said cell indicates the presence or absence ofspecific binding between said first and second proteins of interest. 6.An apparatus according to claim 1, wherein said first and secondproteins are members of a specific binding pair.
 7. A method ofdetecting translocation of a first protein of interest within a cell,comprising: (a) providing a total internal reflection member having asurface portion, with a cell contacted to said surface portion by theplasma membrane of said cell; (b) directing a source light into saidmember to produce an evanescent field adjacent said surface portion,with said evanescent field extending into a first portion of said celladjacent said plasma membrane, said evanescent field being weaker in asecond portion of said cell; wherein said protein of interest has afluorescent group conjugated thereto; said fluorescent group emittinglight when in said first portion of said cell and emitting less lightwhen in said second portion of said cell; and then (c) detecting emittedlight from said fluorescent group, with the emission of more or lesslight from said fluorescent group indicating the translocation of saidfirst protein of interest between said first and second portions of saidcell.
 8. A method according to claim 7, wherein said source light iscoherent light.
 9. A method according to claim 7, wherein said totalinternal reflection member comprises a prism.
 10. A method according toclaim 7, wherein said detecting step is carried out with a CCD camera.11. A method according to claim 7, said cell further containing a secondprotein of interest, said second protein of interest located in eithersaid first portion of said cell or said second portion of said cell,wherein the emission of more or less light from said fluorescent groupindicates the presence or absence of specific binding between said firstand second proteins of interest.
 12. A method according to claim 11,wherein said first and second proteins of interest are members of aspecific binding pair.
 13. A method according to claim 11, furthercomprising the step of administering a test compound to said cell todetermine whether or not said test compound disrupts the binding of saidfirst and second proteins of interest.
 14. A method according to claim13, further comprising the step of repeating steps (a) through (c) atdifferent concentrations of said test compound.
 15. An apparatus forscreening for translocation of a first protein of interest in vivo in aplurality of cells, comprising: (a) a thin unitary total internalreflection member having a surface portion, with (b) a plurality cellcontacted to said surface portion by the plasma membrane of said cell,said cell containing said first protein of interest, said protein ofinterest having a fluorescent group conjugated thereto; (c) a lightsource operatively associated with said total internal reflection memberand positioned for directing a source light into said member to producean evanescent field adjacent said surface portion, with said evanescentfield extending into a first portion of said cell adjacent said plasmamembrane, said evanescent field being weaker in a second portion of saidcell, said fluorescent group emitting light when in said first portionof said cell and emitting less light when in said second portion of saidcell; (d) coupling means for coupling said light source to said thinunitary total internal reflection member and illuminate at least 10square millimeters of said surface portion; and (e) a light detectoroperatively associated with said total internal reflection member andconfigured to detect emitted light from said cells, whereby the emissionof more or less light from said cell indicates the translocation of saidprotein between said first and second portions of said cell.
 16. Anapparatus according to claim 15, wherein said light source comprises acoherent light source.
 17. An apparatus according to claim 15, whereinsaid total internal reflection member comprises a microscope coverslip.18. An apparatus according to claim 15, wherein said light detectorcomprises a CCD camera.
 19. An apparatus according to claim 15 whereinsaid protein having said fluorescent group conjugated thereto is a firstprotein of interest, said cell further containing a second protein ofinterest located in either said first portion of said cell or saidsecond portion of said cell, whereby the emission of more or less lightfrom said cell indicates the presence or absence of specific bindingbetween said first and second proteins of interest.
 20. An apparatusaccording to claim 15, wherein said first and second proteins aremembers of a specific binding pair.
 21. A method of detectingtranslocation of a first protein of interest within a cell, comprising:(a) providing a thin unitary total internal reflection member having asurface portion, with a cell contacted to said surface portion by theplasma membrane of said cell; (b) directing a source light into saidmember through a coupling means to produce an evanescent field adjacentsaid surface portion in an area of at least 10 square millimeters, withsaid evanescent field extending into a first portion of said celladjacent said plasma membrane, said evanescent field being weaker in asecond portion of said cell; wherein said protein of interest has afluorescent group conjugated thereto; said fluorescent group emittinglight when in said first portion of said cell and emitting less lightwhen in said second portion of said cell; and then (c) detecting emittedlight from said fluorescent group, with the emission of more or lesslight from said fluorescent group indicating the translocation of saidfirst protein of interest between said first and second portions of saidcell.
 22. A method according to claim 21, wherein said source light iscoherent light.
 23. A method according to claim 21, wherein said totalinternal reflection member comprises a prism.
 24. A method according toclaim 21, wherein said detecting step is carried out with a CCD camera.25. A method according to claim 21, said cell further containing asecond protein of interest, said second protein of interest located ineither said first portion of said cell or said second portion of saidcell, wherein the emission of more or less light from said fluorescentgroup indicates the presence or absence of specific binding between saidfirst and second proteins of interest.
 26. A method according to claim24, wherein said first and second proteins of interest are members of aspecific binding pair.
 27. A method according to claim 24, furthercomprising the step of administering a test compound to said cell todetermine whether or not said test compound disrupts the binding of saidfirst and second proteins of interest.
 28. A method according to claim26, further comprising the step of repeating steps (a) through (c) atdifferent concentrations of said test compound.
 29. An apparatusaccording to claim 15, wherein said apparatus comprises an invertedwide-field microscope.
 30. An apparatus according to claim 21, whereinsaid apparatus comprises an inverted wide-field microscope.
 31. A methodaccording to claim 28, wherein said thin unitary total internalreflection element is carried by an inverted wide-field microscope.